Bacterial regulatory nucleotides control a wide range of biological processes and their intimate association with persistence and virulence make them promising targets for the development of new drugs. Thus, a thorough understanding of how these nucleotides are produced and of their transducing mechanisms may facilitate the rational design of the next generation of antibiotics. One of the first signaling nucleotide to be identified, ppGpp and pppGpp, collectively known as (p)ppGpp, act as the effector of the stringent response, a stress response that globally reprograms cellular physiology from a ?growth mode? to a ?survival mode?. Despite the efforts of many researchers, our understanding of the mechanisms by which (p)ppGpp promotes bacyerial persistence and virulence is still rather incomplete. This is especially true in Gram-positive bacteria, which does not rely on direct (p)ppGpp-RNAP interactions to globally regulate transcription like Gram-negative bacteria do. Our laboratory has been at the forefront of investigations assessing the genetic and physiologic relevance of (p)ppGpp signaling in Enterococcus faecalis, one of the most common nosocomial pathogens in the Unites States and the rest of the world. The picture that emerged from our studies with E. faecalis is that (p)ppGpp exert differential effects on cell physiology in an incremental manner rather than a biphasic switch between growth and stasis. We found that key phenotypes such as antibiotic persistence and virulence are associated to small fluctuations in (p)ppGpp levels and not to the rapid and drastic accumulation of (p)ppGpp that is observed during the stringent response. Recently, direct evidence of a regulatory loop between (p)ppGpp and the newly discovered c-di-AMP has been demonstrated in both Listeria monocytogenes and Staphylococcus aureus. Although the enzymes that metabolize c-di-AMP are present in the genome of all Enterococci, the functional role of c-di-AMP in this bacterial genus is largely unknown. By demonstrating that c-di-AMP accumulates under (p)ppGpp-inducing conditions, we provided preliminary evidence of a crosstalk between (p)ppGpp and c-di-AMP in E. faecalis. We hypothesize that (p)ppGpp acts in concert with c-di-AMP to regulate bacterial activities that promote stress survival and virulence thereby providing a complementary explanation for how (p)ppGpp modulates bacterial metabolism. The goals of this research plan are to unravel the scope of c-di-AMP regulation in E. faecalis and to determine its relationship with the (p)ppGpp network. Given the generalized role of signaling nucleotides in bacterial pathogenesis, this study is likely to have broad implications and can provide new targets for antimicrobial discovery.